Adamantoate esters of nucleosides



United States Patent Office 3,407,191 Patented Oct. 22, 1968 3,407,191 ADAMANTOATE ESTERS F NUCLEOSIDES Koert Gerzon and Donald L. K. Kan, Indianapolis, Ind., assignors to Eli Lilly and Company, Indianapolis, Ind., a corporation of Indiana No Drawing. Filed Aug. 29, 1966, Ser. No. 575,526 5 Claims. (Cl. 260-2115) ABSTRACT OF THE DISCLOSURE Adamantoate esters of nucleosides are disclosed. The compounds have shown useful activity as anti-viral agents, autolmmune suppressants, and cytotoxic agents.

SUMMARY OF THE INVENTION This invention relates to novel esters'of nucleosides. V

More particularly, it relates to nucleoside adamantoates.

The compounds provided by this invention can be represented by the following formula:

wherein Su is a five carbon sugar residue derived from ribose, arabinose, lyxose, xylose, deoxyribose, or the C and C acetyl derivatives thereof; esterified at C or 0;, with an adamantoyl group,

n Ad-C- wherein Ad represents the adamantyl-l, mono-lower alkyl adamantyl-l, di-lower alkyl adamantyl-l, or tri-lower alkyl adamantyl-l radicals; and substituted at C with B, a nitrogenous base, represented by one of the following formulas:

i N N L and L 2' 0. RIII I r \N wherein R is hydroxy or amino; R is hydrogen, methyl, or halo; Z is CH or N; R is hydroxy, amino, chloro, mercapto, or lower alkyl mercapto; R' is hydrogen, hydroxy, or amino; and Z is t In Formula I above, the free valence on the ring nitrogen is the point of attachment of the sugar. In the part formulas, which Z' in Formula '11 represents, the free valences at each end of the three atom chain are the points of attachment to the pyrimidine ring and the remaining free valence is again the point of attachment of the sugar. The following structural formulas illustrate permissable ring systems coming within the scope of Formula 11 above.

These formulas have been constructed by substituting for Z the above di-radicals which Z' represents.

N R N R OH on q \N q \N N/ L3; N/ Rm \N N/ l l l III IV v R I RI! on NH N \GH N l \N R!!! \N RIII kN v1 vn To further illustrate the nature of the compounds of this invention, the structures of two typical adamantoate esters of nucleosides are set forth below, one containing the pyrimidine ring system of Formula I and the other, the purine ring system of Formula II.

5 O adamantoyl inosine[ 9 (5' O adamantoyl fl- D-ribosyl)hypoxanthine] uracil (a pyrimidine base) xanthine (a purine base) When a nitrogen atom is substituted for a carbon atom in a given pyrimidine or purine, the resulting radical is referred to as an aza compound, with the substituted atom being designated by its numbered position in the pyrimidine or purine ring, as for example 6-azauracil wherein the CH group in the 6 position of the pyrimidine ring is replaced with a nitrogen, or 8-azaguanine wherein the CH group in the 8 position of the purine is replaced with a nitrogen.

The following table gives the names of typically substituted and unsubstituted purines, pyrimidines and related heterocyclic rings exemplary of B in the above formula. In the table, column 1 gives the substituent and column 2 the name of the base thus substituted.

TABLE IContinued Substituent Name of Base R" NH; R' =II Tubcrcidln-base Z=I |ICII=ClI-- (7-deazu-adenine) Rx SH jg i 'Ihloguaniuc.

RZ O H g I 8-azaguanine.

R OH R=I 5-lodouracll. Z CH When D-ribose or D-2-deoxyribose is attached through a p-type linkage to one of the above purine or pyramidine bases, either at the N -position of the pyrimidine ring or at the N -position of the purine ring to form one of the starting materials useful in making the compounds of this invention, the resulting nucleosides have trivial names related to the name of the base from which they are derived. Table II which follows illustrates such trivial names. In the table, column 1 gives the base plus the sugar and column 2 the name of the resulting compound.

TABLE II Base and sugar: Resulting compound Hypoxanthine-D-ribose Inosiue. Adenine-D-ribose Adenosine. 6-mercaptopurine-D-ribose 6-thioinosine. Guanine-D-ribose Guanosine. 7-deaza-adenine-D-ribose 7 deaza adenosine (tubercidin). 6-methylmercap- S methyl 6 thiotopurine-D-ribose inosine. Thymine-D-deoxyribose Thymidine. Uracil-D-ribose Uridine. 5-fluorouracil-D-deoxyribose 5 fiuoro 2' deoxyuridine. 6-azauracil-D-ribose 6-azauradine. Cyt0sine-D-ribose Cytidine. Uracil-D-deoxyribose Deoxyuridine.

Typical compounds coming within the scope of this invention include, but are not limited to, the following:

5-O-adamantoyl-adenosine 5 '-O-( 3 "-methyladam antoyl) -adenosine 5'-O-adamantoyl-inosine 5'-O-adamantoyl-6-thioinosine 5-O-adamantoyl-S-methyl-6-thioinosine 5'-O-adamantoyl-6-chloropurine-riboside (or ribonucleoside) 5-O-ada'mantoyl-6-thioguanosine 5'-O-adamantoyl-2,6-diaminopurine riboside (or ribonucleoside) 5-O-adamantoyl-B-D-arabinosyladenine 5-O-adamantoyl-7-deaza-adenosine (5'-O-adamantolyltubercidin) 5-O-adamantoyl-S-azaguanosine 5'-O-adamantoyl-deoxyadenosine 5-O-adamantoy1-2',3'-diacetyl-adenosine 5'-O-adamant0yl-2-deoxy-S-fiuorouridine 3-O-adamantoyl-2-deoxy-5-fluorouridine 5'-O-adamantoyl-3-acetyl-2'-deoxy-S-fiuorouridine 5'-O-adamantoyl-5-bromo-2'-deoxyuridine 5-O-adamantoyl-2'-deoxy-5-iodouridine 5'-0-adamantoyl-thymidine 5-O-adamantoyl-cytosine-arabinoside 5'-O-adamantoyl-G-azauridine 5 '-O-(3",5 ",7 -trimethyladamantoyl) -6-azauridine and the like.

The compounds of this invention are white crystalline solids. They are prepared by reacting a nucleoside; i.e., a five carbon sugar attached to either a purine or pyrimidine base, with an adamantyl-l-carboxylchloride or anhydride. Because of the fact that the five carbon sugar moiety can react with an acid chloride or anhydride at more than one hydroxyl group, it is customary, if it is desired to prepare an ester on the number 5 carbon, to protect the number 2 and 3 hydroxyls by ketal formation thereon, as with acetone, prior to the reaction with the acylating reagent. In this instance, the acylation reaction is carried out in basic media to avoid hydrolysis of the ketal protective group, which group is readily removed by treatment with acid after the acylation reaction is completed. If the five carbon sugar is a deoxy sugar, however, the hydroxyl group in the number 2 position is not present and ketal formation is not possible; protection of the 3'-hydroxyl by an acetyl group is possible but involves several steps. Fortunately, an acylation reaction with adamantal-l-carboxylchloride carried out on a deoxy nucleoside yields only the 5 adamantoate ester derivative. Other blocking groups can, of course, be used to prevent reaction of the adamantane carboxylchloride or anhydride at the 2' or 3 hydroxyls.

Compounds of this invention in which the adamantoyl group is attached to the sugar through the hydroxyl on the 3 carbon atom are prepared by first blocking the hydroxyl on the 5 carbon with a trityl or like group and then carrying out the acylation reaction in the usual manner. With the 5' position blocked, the adamantyl-lcarboxylchloride reacts with the hydroxyl on the 3' carbon. The desired compound is then readily obtained by removing the blocking group with dilute acid. Compounds of this invention carrying an acetyl group on either one or both of the 2' and 3 positions of the 5 carbon sugar can be prepared by adamantoylation of a nucleoside in which one or more acetyl groups are already present. Alternatively, any of the compounds prepared by the above procedures can be acetylated on both the 2 and 3' positions, in the case of a normal sugar and on the 3 position in the case of a deoxy sugar, by employing acetic anhydride in pyridine or other conventional acetylating agents.

The compounds of this invention are useful for killing virus in vitro as in media suitable for tissue culture experiments. In particular, they are able to suppress the growth of vaccinia virus in a human amnion cell line. They also have considerable cytotoxic activity in vitro and are therefore useful in the study of the relationship of nucleosides to the tumor cell, including the study of the metabolism of nucleosides by tumor cells. It is particularly noteworthy that 5-O-adamantoyl-6-azauridine is quite effective against the ascitic form of the Taper hepatoma in mice, and that 5'-O-adamantoyl-G-thioinosine is quite active against acute lymphatic leukemia strain Ll210 in mice. In fact, this latter compound is more active on a Weight basis than 6-thioinosine itself and has an activity comparable to 6-mercaptopurine, while on a molecular basis it is about 2.5-3 times more active than 6-rnercaptopurine. Furthermore, the compounds of this invention, particularly 5'-O-adamantoyl6-thioinosine, are capable of suppressing antibody formation in mice challenged with sheep erythrocytes. Finally, 5'-O-adamantoyl-adenosine, as well as other compounds of this invention, is capable of inhibiting platelet aggregation and is thus potentially useful as an anticoagulent drug in the treatment of various abnormal cardiovascular conditions.

This invention is further illustrated by the following specific examples.

Example 1.-5'-O-adamantoyl-inosine One gram of 2,3'-O-isopropylideninosine was dissolved in 50 ml. of anhydrous pyridine. 1. 28 grams of adamantane-l-carboxylchloride was then added. The reaction mixture was allowed to remain at ambient room temperature for about 16 hours. Ten milliliters of water were introduced, and the resulting mixture was stirred for about '30 minutes. The volatile materials were removed by evaporation in vacuo. The residue was washed three times with IOO-ml. portions of water, and was then dissolved in ml. of methanol. The methanolic solution was decolorized with activated charcoal. The charcoal was separated by filtration, and the filtrate was evaporated to dryness in vacuo. The resulting residue, comprising 5-O- adamantoyl-2,3'-O-isopropylideninosine, was triturated with ether. This product gave only a single spot on paper strip chromatography using an ethyl acetate-methanol solvent mixture.

The above adamantoyl derivative was dissolved in a mixture of 300 ml. of methanol and 100 ml. of 0.5 N aqueous hydrochloric acid. The resulting solution was heated to reflux for about 45 minutes and then cooled. The acidity of the solution was adjusted to pH=7.0 with cold 10 percent aqueous ammonium hydroxide. The neutralized mixture was decolorized with charcoal and was filtered. Evaporation of the filtrate to dryness yielded 5'- O-adamantoyl-inosine, which melted in the range 202- 205" C. after recrystallization from an ethyl acetatemethanol solvent mixture. This compound also yielded only a single spot on paper strip chromatography.

Analysis.-Calc.: C, 58.59; H, 6.09; N, 13.02. Found: C, 58.53; H, 6.33; N, 12.44.

5-0-(3",5"-dimethyladamantoyl)inosine was prepared by the above procedure except that 3,5-dimethyladamantane-l-carboxylchloride was substituted for adamantanel-carboxylchloride. 5'-O-(3,5"-dimethyladamantoy1)inosine thus prepared was isolated and purified by the above procedure and melted at 208209 C. after recrystallization from an ethyl acetate-methanol solvent mixture.

Analysis.Calc.: C, 60.25; H, 6.59; N, 12.22. Found: C, 60.10; H, 6.75; N, 12.46.

5-O-adamantoy-l-adenosine was prepared by the above procedure except that isopropylidenadenosine was used in place of isopropylideninosine. 5'-O-adamantoyl-adenosine thus prepared was recrystallized from dichloromethane.

Analysis.--Calc.: C, 58.73; H, 6.34. Found: C, 58.66; H, 6.75.

5' O adamantoyl 6 thioinosine was prepared by the above procedure except that isopropylidene-6-thioinosine [prepared by the procedure of Zderic, Moffat, Gerzon, Kau and Fitzgibbon, J. Med. Chem., 8, 275 1965)] was used in place of isopropylideninosine. 5'-O-adamantoyl- 6-thioinosine thus prepared was recrystallized from methanol. M.P.=226-227 C.

Analysis.- Calc.: C, 56.48; H, 5.87; N, 12.55. Found: C, 56.59; H, 6.08; N, 12.48.

Other compounds prepared by the above procedure include: 5 O (3",5" dimethyladamantoyl)adenosine; M.P.=l58159 C.

Analysis-Cale: C, 60.38; H, 6.83; N, 15.31. Found: c, 60.23; H, 6.85; N, 15.05.

5' O (3",5",7 trimethyladamantoyl)adenosine; M.P.=179-180 C.

Analysis.-Ca'lc.: C, 61.13; H, 7.05; N, 14.85. Found: C, 60.89; H, 7.46; N, 14.43.

5' O (3", 5", 7" trimethyladamantoyl) 6 azauridine; M.P.=122-124 C.

Analysis.Calc.: C, 58.78; H, 6.95; N, 9.35. Found: C, 58.76; H, 7.18; N, 9.69.

5 O adamantoyl 6 azauridine; M.P.=179 180 C.

Analysis.-Calc.: C, 56.01; H, 6.19; N, 10.31. Found: C, 55.95; H, 6.34; N, 10.06.

5' O (3",5" dimethyladamantoyl) 6 azauridine; this compound did not crystallize but was proved to be one spot material by thin layer chromatography on silica gel in a chloroform-ethyl acetate solvent mixture.

Example 2.'-O-adamantoyl-2'deoxyadenosine Following the procedure of Example 1, a solution of adamantane l carboxylchloride in benzene was added dropwise to a solution of 2'-deoxyadenosine in anhydrous pyridine. The reaction mixture was allowed to remain at ambient temperature for 16 hours after which time it was demonstrated by thin layer chromatography that all the starting 2'-deoxyadenosine had reacted. Fixe milliliters of water were added. The resulting mixture was stirred for 30 minutes, and then was concentrated in vacuo. The resulting residue comprising 5 O adamantoyl 2' deoxyadenosine was dissolved in ethyl acetate. The ethyl acetate layer was washed successively with water, percent sodium bicarbonate solution, and water. Evaporation of the solvent yielded a residue of 5-O-adamanatoyl- 2'-dioxyadenosine which was crystallized from a benzenedichloromethane solvent mixture. 5 O adamantoyl- 2-deoxyadenosine thus prepared melted at about 182- 183 C.

Analysis.-Calc.: C, 61.00; H, 6.58; N, 19.94. Found: C, 61.23; H, 6.56; N, 16.75.

Following the above procedure 2'-deoxy-5-iodouridine was reacted with adamantane-l-carboxylchloride to yield 5' O adamantoyl 2' deoxy 5 iodouridine. Thin layer chromatography showed that the noncrystalline product of this reaction was a single substance.

Other compounds prepared by the above procedure include: 5' O adamantoyl 2' deoxy 5 fluorouridine; M.P.=116117 C.

Analysis.Calc.: C, 58.81; H, 6.17; N, 6.86. Found: C, 58.51; H, 6.29; N, 6.89.

5-O-adamantoyl-thymidine; M.P.=l90191 C.

AnaIysis.--Cal.: C, 62.34; H, 7.05; N, 6.92. Found: C, 62.42; H, 7.11; N, 7.21.

Example 3.3'-O-adamantoyl-2'- deoxy-S-fiuorouridine To a solution of 50 mg. of 2-deoxy-5-fluoro-5'-O-trityluridine [prepared by the method of Thomas and Montgomery, J. Med. Chem., 5, 24 (1962)] in 5 ml. of anhydrous pyridine was added 50 mg. of adamantane-lcarboxylchloride. The resulting reaction mixture was stirred for about 16 hours at ambient temperature. Two milliliters of water were added, and the mixture evaporated to dryness in vacuo. The residue was washed twice with 10-ml. portions of water and was then dissolved in 50 ml. of chloroform. The chloroform solution was washed with water and dried. Evaporation of the solvent yielded 3' O -adamantoyl 2' deoxy 5 fluoro 5- O-trityluridine.

The residue was dissolved in 50 ml. of 80 percent acetic acid. This solution was refluxed for about 20 minutes, cooled, and then evaporated to dryness in vacuo at a temperature of about 4550 C. The resulting residue was washed twice with 10-ml. portions of water, and the last trace of the water was removed by evaporation in vacuo. The residue, containing 3-O-adamantoyl-2-deoxy-5-fiuorouridine, was crystallized from hexane and melted at about 209210 C.

Analysis.-Calc.: C, 58.81; H, 6.17; N, 6.86. Found: C, 58.74; H, 6.10; N, 6.81.

Example 4.5'-O-adamantoyl-2-deoxy-5-fluorouridine from 3'-O-acetyl-2-deoxy-S-fluorouridine 5' O adamantoyl 2 deoxy 5 fluorouridine was prepared from 3' O acetyl 2 deoxy 5 fiuorouridine described by Thomas and Montgomery (loc. cit.) in the following manner: Two hundred milligrams of adamantane-l-carboxylchloride were added to a stirred solution containing 270 mg, of 3-O-acetyl-2'-deoxy-5-fluorouridine in ml. of anhydrous pyridine. After 16 hours, 5 ml. of Water were added, and the mixture was stirred for minutes. The mixture was concentrated in vacuo,

and the resulting residue was dissolved in ml. of chloroform. The organic layer was washed with water and dried. The solvents were removed by evaporation in vacuo. Trituration of the resulting residue with hexane yielded solid material. Recrystallization of the solid from an ether cyclohexane solvent mixture yielded 3'- acetyl 5' adamantoyl 2' deoxy 5 fluorouridine: M.P.=6265 C.

Analysis.Calc.: N, 6.22. Found: N, 6.19.

Preferential hydrolysis of 3 acetyl 5' adamantoyl- 2' deoxy 5 fluorouridine was accomplished by dissolving the compound in 0.25 N sodium hydroxide in aqueous dioxane (1:1), and stirring the resulting solution for about 20 minutes. 5' adamantoyl 2- deoxy-5-fiuorouridine thus prepared was isolated by standard procedures and proved to be identical in all respects with the material obtained by the procedure of Example 2.

Example 5.5'-O-adamantoyl-2',3-diacetyladenosine Two milliliters of acetic anhydride were added to a solution containing .52 g. of 5'-O-adamantoyl-adenosine in 5 ml. of pyridine. The reaction mixture was maintained below 30 C. for about 4 hours. Five milliliters of ethanol were introduced, the mixture was stirred for 30 minutes, and then concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate, and the ethyl acetate layer was washed with cold, dilute sodium bicarbonate solution and again with water. The solution was dried and concentrated in vacuo. The residue thus obtained was dissolved in ether. Addition of hexane caused an amorphous solid to precipitate. The precipitate was filtered, and the filter cake was recrystallized from ethyl acetate. 5'-O-adamantoyl-2,3'-diacetyladenosine thus prepared melted at about 236237 C.

Analysis-Cale; N, 13.82. Found: N, 13.82.

Example 6.Biological evaluation I 66 percent activity 30 mg./kg. of mouse body weight for ten days intraperitoneally.

(B) Leukemia L121O in mice 30-48 percent activity 30 mg./kg. of mouse body weight for ten days intraperitoneally.

(C) Antibody formation in mice after sheep erythrocyte injection percent activity 300 mg./kg. mouse body weight-one injection postantigen.

(2) 5'-O-adamantoyl-6 azauridine (A) Taper hepatoma ascites in mice 63-67 percent activity 500 mg./kg. of mouse body weight for ten days intraperitoneally.

dine (A) Taper hepatoma ascites in mice 5'-O-(3"', 5 7-trimethyladamantoyl)-6-azauri- 3242 percent activity 300-400 mg./ kg. mouse body weight for ten days intraperitoneally.

We claim: 1. A compound of the formula wherein Su is a five carbon sugar residue derived from ribose, arabinose, lyxose, xylose, deoxyribose, or the C and C acetyl derivatives thereof; esterified at C or C with an adamantoyl group,

O ltd-L wherein Ad represents the adamantyl-l, mono-lower alkyl adamantyl-l, di-lower alkyl adamantyl-l, or trilower alkyl adamantyl-l radicals; and substituted at C with B, a nitrogenous base, represented by one of the following formulas:

I wherein R is hydroxy 01' amino; R is hydrogen, methyl, or halo; Z is CH or N; R" is hydroxy, amino, chloro, mercapto, or lower alkyl mercapto; R' is hydrogen, hydroxy, or amino; and Z is References Cited UNITED STATES PATENTS 7/1953 Holly et a]. 260211.5 9/1967 Anraku et al. 260-2115 LEWIS GOTTS, Primary Examiner. v

JOHNNIE R. BROWN, Assistant Examiner. 

